The present invention relates to methods of manipulating molecules and, in particular, to a fluid transport system useful for straightening, aligning, and fixing long chain polymers such as DNA.
The analysis of nucleic acid molecules (e.g. DNA) and, in particular, the sequencing of such molecules may be aided by optical techniques in which long portions of such molecules are straightened and fixed to a substrate for microscopic analysis. The fixed molecule may be analyzed by creating “landmarks” on the molecule by attaching fluorescent markers to specific locations or by cutting it with restriction enzymes to form visible breaks at specific locations. The order and relative separation of the landmarks is preserved, because the molecule remains fixed, and may be used to produce an optical map of the molecule. The optical map provides a framework on which other sequence information may be assembled. The landmarks allow optical maps of fragments of long molecules to be assembled into the entire molecule by the process of matching fragments with similarly spaced landmarks.
The effective use of optical maps requires that large numbers of single molecules be processed. A number of techniques have been examined for the purpose of straightening and fixing large numbers of molecules including: (1) straightening the molecules in a flow of molten gel which is then hardened to fix the molecules in place and (2) straightening the molecules under capillary flow of a carrier liquid or convective flow caused by evaporation of a carrier liquid and promoting adsorption of the elongated molecules to a substrate adjacent to the flow.
A different set of techniques has been investigated in which the molecules are straightened in a flowing carrier fluid without being fixed to a substrate. In these techniques, the molecules are analyzed as they move. While these latter techniques potentially provide the same benefits of preserving the order and relative separation of the landmarks, motion of the molecule complicates the process of imaging the molecule, makes some landmarking techniques difficult, and eliminates the possibility of preserving the molecule for later additional or more complex analysis.
Ideally, when molecules are fixed to a substrate, the fixed molecules should have sufficient separation so that molecules do not overlap or cross. Points of overlap create image artifacts that can severely hamper the analysis process.
It is typical to stain the fixed molecule with a fluorescent material which distributes itself evenly along the molecule allowing estimates of separation between landmarks (e.g., in numbers of base pairs) to be gauged by total fluorescence rather than strictly by length. Such fluorescence measurements work best if the elongation of the molecule during straightening is not so great as to decrease the fluorescence per length of the molecule to a background level. Inadequate elongation of the molecule, however, can make it difficult to identify the points cut by the restriction enzymes, which desirably separate slightly under relaxation of the elongated molecule to render the cuts visible.
Prior art techniques for elongating and fixing long chain molecules can produce excessive overlap among molecules and variation in molecule elongation.